The term chromatography embraces a family of closely related separation methods. The feature distinguishing chromatography from most other physical and chemical methods of separation is that two mutually immiscible phases are brought into contact wherein one phase is stationary and the other mobile. The sample mixture, introduced into the mobile phase, undergoes a series of interactions (partitions) many times before the stationary and mobile phases as it is being carried through the system by the mobile phase. Interactions exploit differences in the physical or chemical properties of the components in the sample. These differences govern the rate of migration of the individual components under the influence of a mobile phase moving through a column containing the stationary phase. Separated components emerge in the order of increasing interaction with the stationary phase. The least retarded component elutes first, the most strongly retained material elutes last. Separation is obtained when one component is retarded sufficiently to prevent overlap with the zone of an adjacent solute as sample components elute from the column.
WO 9729825 (Amersham Pharmacia Biotech AB) discloses one kind of chromatography, wherein mixed mode anion exchangers provide interactions based on charges and hydrogen-bonding involving oxygen and amino nitrogen on 2-3 carbons' distance from positively charged amine nitrogen. The chromatography is based on the discovery that this kind of ligands can give anion exchangers that require relatively high ionic strengths for eluting bound substances.
More recently, a kind of ligands denoted high salt ligands was disclosed, see e.g. WO 01/38227 (Amersham Biosciences AB, Uppsala, Sweden). These ligands can act as mixed mode anion-exchange ligands, and have shown to be of great interest in many industrial applications, such as protein purification, since they can withstand high salt concentrations and accordingly does not require any substantial dilution of the sample. Thus, the high salt ligands are advantageously used for biotechnological separations, since they reduce the total volume of sample required as compared to previously described methods, and accordingly reduce the total cost for equipment as well as work effort required in such applications.
However, even though the mixed mode anion-exchange ligands reduce costs and efforts when used in separation, the hitherto described methods for the preparation thereof involves certain drawbacks that have made them less advantageous in practice. In most cases, the immobilisation of these mixed mode anion exchanger ligands is based on opening of an epoxide-derivatised gel by the amine groups of the ligand. Since this kind of nucleophilic substitutions are very dependent on the pKa of the amine group, but also of its nucleophilicity and of steric hindrance factors, no general method can be applied and optimisation of the immobilisation has to be performed for each specific case. Furthermore, in the case of amine groups with poorer nucleophilicity, a large excess (more than two equivalents) of expensive ligands were required. Another drawback is that due to the basic difference of reactivity, large differencies in the conditions of the immobilisation have to be optimised, e.g. between secondary and tertiary amines.
Another drawback of the above-mentioned WO 01/38227 is that since the immobilisation is performed via the amine function, it will not be possible to directly obtain media that contain primary amine groups.
To obtain primary amine groups, the authors of the above-mentioned WO 01/38227 have used some protective groups, which make the production longer and hence more costly, and also increase the risk to yield non-homogenous media due to incomplete deprotection.
Another solution to generate primary amines on the media is demonstrated by the use of polyamines. However, these polyamines can be attached by multiple points and again result in media with poor homogeneity.
Accordingly, none of the suggested methods are general and reliable enough to be used in the generation of libraries or in a parallel format.
A specific solution to the similar problems that are known in relation to the preparation of mixed mode cation-exchange ligands has been suggested in PCT/SE02/01650 (Amersham Pharmacia Biotech, Uppsala, Sweden), which application however was not public at the time of the filing of the present application. Disclosed is a three functional scaffold, preferably homocysteine thiolactone, which is used as a starting material for the preparation of cation-exchange ligands. The method allows generating a library of various ligands with great diversity.
Since at present there are no available functional alternatives to the method described above, there is a need within this field of improved methods for the manufacture of mixed mode anion-exchange ligands for use in separation procedures.
Finally, Feist and Danna (“Sulfhydryl cellulose: A New Medium for Chromatography of Mercurated Polynucleotides”. Patricia L. Feist and Kathleen J. Danna, Biochemistry, 20(15), p. 4243-4246) have disclosed a process of preparing sulfhydryl cellulose, which process includes to mix amino ethyl cellulose with an N-acetylhomocysteine thiolactone.